The intestine is a vital part of the gastrointestinal tract (GIT) that primarily functions to absorb nutrients and water from ingested food and drinks. In consequence, it is constantly exposed to digestive juices, containing enzymes and other agents that act to break down nutrients, dietary antigens and potentially pathogenic microorganisms from the external environment. In addition, the intestine houses the gut flora, myriads of microorganisms from about 500 different species, including bacteria, archaea and eukaryotes.
The mucosa as the innermost layer of the GIT is the interface that is exposed to and interacts with the external environment and the luminal content of the intestine. It consists of epithelial cells forming crypts and villi, subepithelial tissue and lymph nodes (lamina propria), and underneath a continuous sheet of smooth muscle cells (muscularis mucosae). The entire mucosa rests on the submucosa, which consists of a variety of inflammatory cells, lymphatics, autonomic nerve fibers, and ganglion cells. The mucus layer lining the mucosal epithelial cells is the first defensive barrier that protects the underlying mucosa from the entrance of harmful substances or pathogens. Mucus is a complex viscous composition, which typically forms layers surrounding the intestinal lumen. It consists of large glycoproteins, called mucins, which are secreted by specialized epithelial cells and serve as a scaffold for the mucus gel, further containing salts, lipids and proteins (Johansson, et al., 2011) (Turner, 2009). The mucus layer converts the hydrophilic epithelial surface into a hydrophobic “closing seal” that interfaces with luminal contents (Hicks, et al., 2006) (Hills, 2002). Phosphatidylcholines (PC) bind to the negatively charged mucins with their positively charged headgroups while their hydrophobic acyl chains extend to the lumen, thus establishing a high surface tension which normally helps to exclude bacteria from this compartment (Willumeit, et al., 2007) (Guo, et al., 1993).
However, under certain circumstances, bacteria somehow overcome the mucosal barrier of the intestine and elicit intestinal inflammation. Inflammatory bacterial diarrhea is a significant health problem in both developing and developed regions of the world that particularly affects children, elderly persons, and immunosuppressed individuals. The standard treatment is mostly restricted to antibiotics. However, antibiotic therapy is often leveraged by bacterial antibiotic resistances. In addition, antibiotics often elicit severe side effects and can unbalance the gut flora, which may lead to follow-up infections caused by other pathogenic microorganisms.
Intestinal inflammation is also a characteristic of inflammatory bowel diseases (IBD). These chronic, relapsing diseases have been linked to a dysregulated immune response to components of the gut flora. IBD have therefore long been classified as “autoimmune” diseases and are typically treated with anti-inflammatory agents, e.g., corticosteroids, immunosuppressives, antibiotics or even surgical approaches in those who are non-responders to medical treatment. The disadvantages of antibiotic treatment have already been elucidated, and many anti-inflammatory and immunosuppressive agents, too, evoke severe side effects. Surgery should, of course, be the last resort for treatment of IBD (Triantafillidis, et al., 2011).
The exact mechanisms whereby bacteria disrupt and enter the intestinal mucus and the underlying mucosa are still the subject of ongoing research. Despite the differences regarding disease etiology and pathogenesis, the present inventors have identified one common factor shared by many inflammatory intestinal diseases: Bacteria or bacterial antigens cross the natural mucosal barrier and reach the underlying mucosa, where an inflammatory response arises. This understanding lead to the idea to implement treatment at the beginning, i.e. by preventing bacterial invasion of the mucus barrier. And, for the first time, it is herein suggested to do so by inhibiting bacterial phospholipase activity.
Phospholipases (PL) are abundant throughout the prokaryotic and eukaryotic kingdom and constitute a heterogenous group of diverse lipolytic enzymes that share the ability to hydrolyze one or more ester linkages in phospholipids. PL are typically classified based on their site of action; whether they cleave in the hydrophobic diacylglycerol moiety (PLA) or in the polar head group of the amphipathic phospholipid (PLC and PLD). PLAs can be further defined by their positional specificity, i.e. preference for the acyl group attached to position 1 or 2 of the glycerol backbone, as PLA1 and PLA2, respectively; PLBs have both PLA1 and PLA2 activity, i.e., little or no positional specificity (Istivan & Coloe, 2006).
Relatively few studies have elucidated the role of bacterial PL, in particular bacterial PLA2, in host-pathogen interactions. It has been acknowledged that the action of bacterial PLA2 results in the accumulation of free fatty acids and lysophospholipids, which are known to destabilize (host) membranes. Few data suggesting a role in pathogenesis for bacterial PLA, including PLA from Vibrio parahaemolytics, Ricksettia prowazekii and Campylobacter coli, have been linked to hemolytic activity due to the accumulation of lysophospholipids (Schmiel & Miller, 1999) (Istivan & Coloe, 2006). It has further been speculated that bacterial PLA might promote bacterial survival and growth by disrupting innate immune cells, thereby hampering the host's immune defenses, and by providing nutrients in the form of fatty acids for biosynthesis or metabolism. Another option raised was that Yersinia enterocolitica PLA may stimulate the pro-inflammatory arachnidonic acid cascade by releasing fatty acids, including arachidonic acid, from the glycerol backbone of phospholipids (Schmiel & Miller, 1999). Other studies gave a contradictory picture for bacterial PLA. For example, the injection of Salmonella newport into ligated ileal loops induced similar levels of fluid accumulation, desquamation, and mononuclear cell infiltration as the injection of bacteria. In contrast, a PLA mutant strain of Vibrio cholerae was reported to induce similar amounts of fluid accumulation in rabbit ligated ileal loops compared to the parent strain. (for review, see (Schmiel & Miller, 1999) (Istivan & Coloe, 2006)).
Even though antibiotics, anti-inflammatory, immunosuppressive and anti-diarrheal agents are available for the treatment of inflammatory bacterial diseases of the intestine, there is still a need for alternative or additional drugs for combating such diseases, because of bacterial resistances to antibiotics, the potential damage of the host's gut flora and the adverse effects.
The technical problem underlying the present invention is to comply with this need. The solution is set out in the claims and the description in aspects and embodiments of the present invention that follow as well as illustrated by the figures and exemplified in the appended examples. To date no therapeutics targeting the detrimental effects of bacterial PLA in diseases affecting the intestine have been developed; as their role of has largely been ignored.
The present inventors, for the first time, present an approach to treat various inflammatory bacterial diseases of the intestine by inhibiting bacterial phospholipases (PL), in particular bacterial PLA2. Of note, therapeutic approaches targeting PLA in inflammatory diseases, and i.a. inflammatory bowel diseases, have focused on inhibiting endogenous host PLA—presumably because host PLA are implicated in a variety of signaling pathways and mechanisms; many of which are of relevance in the pathogenesis of inflammatory diseases (see (Linkous & Yazlovitskaya, 2010), (Murakami, et al., 2011)). For example, one function of host soluble PLA2 (sPLA2) is antimicrobial defense through degradation of bacterial membrane phospholipids (see (Murakami, et al., 2011) for review). An increased host cytoplasmic PLA2 (cPLA2) activity has also been reported in tissues infected with Mycobacterium tuberculosis, Pseudomonas aeruginosa, Listeria monocytogenes and Helicobacter pylori (see (Linkous & Yazlovitskaya, 2010) for review).
Accordingly, several host PLA2 inhibitors have been developed to inhibit or decrease host PLA2 activity. For example, WO9944604 provides an inhibitor of human non-pancreatic sPLA2 for the treatment of inflammatory diseases, including inflammatory bowel diseases. The approach however ignores the role of gut bacteria implicated in IBD, and provides an anti-inflammatory agent to disrupt the host's immune overreaction to the gut flora, rather than targeting harmful effects of the gut flora itself. Similar approaches have been adopted by, e.g., US2005075345, GB2346885, EP0465913, U.S. Pat. No. 6,110,933.
Interfering with host PLA2 activity, however, poses risks. Host PLA2 are abundant and fulfill a plethora of functions. Hence, drugs inhibiting host PLA2 activity are predestined for eliciting systemic side effects, e.g. by impeding membrane maintenance, signaling and immune defense mechanisms. In fact, targeting host PLA2 may even exacerbate bacterial intestinal diseases, as host secreted PLA2 reportedly participate in host defense by destroying bacterial membranes. Instead of providing just another anti-inflammatory agent that attenuates the host's response to bacterial challenge, the present inventors were the first to specifically target bacterial PL, in particular bacterial PLA2 activity of harmful bacteria, thereby avoiding an impairment of host PLA function. This clearly renders the approach superior to state-of-the-art therapeutics for the treatment of inflammatory bacterial diseases of the intestine.
In contrast to any other approach revealed in the prior art, the present inventors have developed a way to effectively target a key event in the pathogenesis of inflammatory bacterial diseases of the intestine: the invasion of the protective intestinal mucus barrier. As of this writing, efforts have been put into inhibiting host PLA to decrease inflammation and tissue destruction in a variety of inflammatory diseases. The present inventors were the first to understand that bacterial PL, in particular bacterial PLA2, are crucial for the pathogenesis of a vast number of inflammatory bacterial diseases affecting the intestine, and, importantly, that inhibiting bacterial PL, in particular bacterial PLA2, activity offers an elegant solution to the task of providing a therapy that acts adversely neither on the patient, nor on his gut flora.
In addition, the present invention provides lysophospholipid-conjugates as inhibitors of bacterial PL, in particular bacterial PLA2, and, consequently, as potent therapeutics for the treatment of a vast number of inflammatory bacterial diseases of the intestine. Clearly, the ability of lysophospholipid-conjugates to interfere with bacterial PLA could not be foreseen.
UDCA-LPE, being one exemplary lysophospholipid-conjugate according to the present invention, had initially been designed for the delivery of the phospholipid-precursor LPE to the steatotic liver, which typically exhibits low PC/LCP levels that have been linked to an increased PLA2 activity (Chamulitrat, et al., 2009). The hepatoprotective effects of UDCA-LPE have been linked to the inhibition of hepatic PLA2 which, on the one hand, results in disintegration of the fatty acid uptake complex and, on the other hand, suppression of the cytosolic generation of lysophosphatidylcholine (LPC), which in turn results in deactivation of JNK1, a common promoter of fatty acid influx, inflammation and apoptosis (Stremmel & Staffer, 2012) (Stremmel, et al., 2012). Of note, phospholipases are a very diverse group of enzymes. For example, although all, eukaryotic and prokaryotic, sPLA2s share the same catalytic mechanism, there is considerable variation in their sequence identity and structure (Nevalainen, et al., 2012). Hence, the finding that UDCA-LPE interferes with hepatic PLA2 can certainly not be expanded to bacterial PLA. The finding that lysophospholipid-conjugates can effectively decrease bacterial PLA activity and therefore exhibit a significant potential for the treatment of intestinal diseases associated with inflammation and, eventually, bacterial invasion of the mucosal barrier of the intestine, therefore clearly came as a surprise. The present invention offers a new, unexpected way to treat inflammatory intestinal diseases that acts more specific, is less toxic and more convenient than state-of-the-art methods.